1. Fields of the Invention
The present invention relates to a method for preparing a precursor used for labeling hepatocyte receptors, especially to a method for preparing a precursor used for labeling hepatocyte receptors and containing bifunctional structure including trisaccharide and DTPA ligand.
2. Descriptions of Related Art
There are no nerves in the liver. Thus most people with liver diseases have no feelings or specific symptoms until their liver disease is at a late stage. Once there are some symptoms and people go to hospital for diagnosis, they already have serious hepatocirrhosis and liver cancer. Thus early diagnosis and treatment of liver diseases are so important to saving lives. Early detection of liver fibrosis will benefit the prevention of the liver diseases. Thus the early detection of liver diseases has received an enormous amount of attention in medical research.
The most common test for liver fibrosis is to get liver biopsy through liver puncture. This is an invasive test associated with the risk of sampling error and morbidity. Thus non-invasive tests to assess liver fibrosis have been developed and proposed by various research teams and institutions to assess the severity of hepatic fibrosis. One of the most promising ways is to use isotope tracer technique for detecting diseases or functional disorders. The isotope tracer technique is safe, painless (non-invasive), convenient and accurate. Moreover, due to fast development of techniques for molecular images, Positron Emission Tomography and Single-photon Emission Computed Tomography (SPECT) can provide functional diagnosis. In recent years, computed tomography (CT) and magnetic resonance imaging (MRI) are also used to overcome shortcomings of PET or SPECT on anatomical imaging.
Human cells have specific receptors on their surfaces to accept specific proteins or peptides. According to the specificity, some specific proteins or peptides are labeled with radioactive nuclides in advance and then delivered into human bodies. The labeled proteins or peptides will achieve higher concentration in specific organs or tissues so as to treat diseases or for diagnosis.
There are about two hundred thousand asialoglycoprotein receptors (ASGPR) on surfaces of mammalian hepatocytes. The ASGPR also has high affinity to galactose (Gal) and N-acetylgalactosamine (GalNAc). Especially ground substances that contain tri-Gals or substrate with tri-GalNAc have a high affinity for ASGPR on surfaces of hepatocytes, almost 106 times of a single saccharide. Moreover, the affinity varies due to the length of a single chain. For example, the affinity of DCM-Lys(G-ah-GalNAc)3 to hepatocytes is 12 times than the affinity of DCM-Lys(ah-GalNAc)3 to hepatocytes even there is only a difference of a Glycine. In accordance with the above affinity and specificity, not only the drug targeting is improved, the amount of the drug used is minimized to nM level. Furthermore, based on the above biomedical character, Glyco-drugs are labeled with radioactive isotopes. Used together with nuclear imaging machines, a non-invasive and quantitative liver function test technique is available and this is beneficial to prevention and treatment of liver diseases.
In some papers, a method for synthesis of DTPA-aha-DCM-Lys(G-ah-GalNAc)3 is revealed. But the synthesis processes are complicated and time-consuming. Refer to FIG. 1, during the synthesis, the trisaccharide skeleton is obtained by Nε-benzyloxycarbonyl-L-lysine and bromoacetic acid reacting in sodium hydroxide solution. However, it is not assured that amino group is exactly connected to two carboxylic acids. If there is only one carboxyl acid group connected, disaccharide is formed in following synthesis processes, instead of trisaccharide.
Moreover, there are two times of Sephadex gel chromatography such as Sephadex LH20 or Sephadex G-15 during the synthesis and the cost is expensive. Alcohol or acetic acid solution is used as elution solution for purification. The concentration time is increased due to the acetic acid solution. Moreover, hydroxide ion exchange resin is used to remove impurities completely. It's time and cost consuming are not suitable for general laboratories.
Furthermore, after purification, the product is filtered several times and then is crystallized to remove impurities. Yet the solvent is N,N-dimethylformamide (DMF) with high boiling point and the coupling agents including N,N′-dicyclohexyl-carbodiimide (DCC) and N-Hydroxybenzotrizole (HOBt). These are all difficult to be removed completely by filtering or recrystallization. This causes troubles to the next process.